The most frequent cause of death in our country is malignant neoplasm, and in particular, the mortality of lung cancer in men is the highest, exceeding that of stomach cancer, while the mortality is the third highest in women and tends to be increasing every year. Lung cancer is histopathologically classified into the following four main subtypes, namely, squamous-cell carcinoma and small-cell lung carcinoma (SCLC) occurring in the hilar region, adenocarcinoma and large-cell lung carcinoma occurring in the lung field.
In particular, since small-cell lung carcinoma proliferates fast and causes remote metastasis in the early stage, it is often discovered, even on the initial diagnosis, to be progressive cancer in which metastasis has already occurred systemically. The cure rate of this type of cancer is approximately 20% for the patients with the limited disease (LD) type of small-cell lung carcinoma, in which pathological lesion is limited only to one side of the lung; however, for the patients with the extensive disease (ED) type in which cancer has metastasized to both lungs or to other organs, complete cure is said to be practically difficult.
Furthermore, since small-cell lung carcinoma is highly sensitive to anticancer drugs, chemotherapy is considered to be the best choice for the treatment of the disease. However, the rate of success with chemotherapy is low for non-small-cell lung carcinoma (non-SCLC), and thus surgical treatment is known to be the best choice for the treatment.
Therefore, small-cell lung carcinoma is a cancer which particularly necessitates early discovery and early treatment among lung cancers, and for this reason, differential diagnosis of small-cell lung carcinoma and non-small-cell lung carcinoma is extremely important for making decision on the course of treatment.
One of the methods for discovering lung cancer is sputum examination. However, although sputum examination is suitable predominantly for the examination of squamous-cell carcinoma, there is a problem that the positive rate for small-cell lung carcinoma is low. Also, X-ray imaging is another method widely used in the discovery of lung cancer; however, with regard to squamous-cell carcinoma or small-cell lung carcinoma which occurs in the hilar region, there is a problem that imaging of the shadow of cancerous tissues is very difficult because the shadow of the heart falls on the hilar region. Furthermore, with regard to small-cell lung carcinoma, it is believed that even though those patients who show anomalous shadow of the lung field are diagnosed using sputum cytodiagnosis, simple chest X-ray imaging, CT scanning, bronchoscopy and the like, early discovery of this type of lung cancer is never easy.
In addition, several examinations for diagnosing cancer, such as irradiation, biopsy and bronchoscopy, cause pain in patients, and require expensive instruments or skilled engineer.
Therefore, research is being conducted to find a tumor marker which enables highly efficient diagnosis of cancer at a curable stage through a more convenient blood examination method. Currently, 30 or more tumor markers are being used in the discovery and diagnosis of cancer patients, indication for monitoring of the course of disease, diagnosis of recurrence, or the like.
Since lung cancers have various subtypes, there is no report on a tumor marker which is effective in the discovery or diagnosis of all subtypes of lung cancer. Thus, at the present, effective tumor markers are selected and used in accordance with each subtype of lung cancer.
For example, carcinoembryonic antigen (CEA) or sialyl Lex-i antigen is mainly chosen and used for pulmonary adenocarcinoma, squamous-cell carcinoma related antigen (SCC) for squamous-cell carcinoma, and neuron-specific enolase (NSE) for small-cell lung carcinoma.
However, NSE is disadvantageous in that (1) the positive rate is low at an early, curable stage in SCLC; (2) a transient increase in the measured values is recognized upon treatment; (3) the measured values increase due to hemolysis during blood collection; (4) the difference in the measured values between small-cell lung carcinoma patients and normal persons is small; and the like. Thus, NSE could not necessarily be said to be an effective tumor marker for small-cell lung carcinoma.
Gastrin-releasing peptide (GRP) is a brain gut peptide comprising 27 amino acids, which was isolated from porcine stomach tissues by McDonald et al. in 1978, and has a gastrin secretion promoting effect. The presence of GRP in human has also been confirmed, and the gene encoding human GRP has been also cloned in 1984.
Yamaguchi et al. at the National Cancer Center in Japan measured 15 or more types of brain gut hormones, including adrenocorticotropic hormone (ACTH), calcitonin and the like, in the course of investigating the biological characteristics of small-cell lung carcinoma, which is conceived to be derived from neuroendocrine cells, and clarified that GRP is actively secreted from cultured small-cell lung carcinoma cell lines at the highest frequency and highest concentration (Non-Patent Document 1). Moreover, they also established a radioimmunoassay (RIA) combined with a method of concentrating GRP in blood, and found that patients with small-cell lung carcinoma would show higher blood concentration of GRP compared to normal persons. However, since GRP is rapidly digested in the blood, its concentration in blood is low, and since the aforementioned assay requires complicated concentration processes, clinical application is difficult.
From researches conducted thereafter, it was found that three species of GRP precursors (proGRP) are produced by alternative RNA splicing in various cells (Non-Patent Document 2). These three species of ProGRP show that the 1st to 98th amino acids in the amino acid sequence are common, while the amino acid sequence on and after the 99th amino acid is different from each other, due to alternative RNA splicing. This common portion in the amino acid sequence of from the 1st to 98th amino acids, is shown in SEQ ID NO:1. Hereinafter, unless stated otherwise in particular, the numbering of amino acid residues in ProGRP according to the present invention, partial amino acid sequences thereof, digests and the like, is based on the numbering of the amino acid sequence of SEQ ID NO:1.
The amino acid sequence of from the 1st to 27th amino acids in the three species of ProGRP is identical to the amino acid sequence of mature GRP having gastrin secretion promoting activity. These three species of precursors are all digested by hormone precursor cleavage enzymes, into mature type GRP having an amino acid sequence consisting of amino acids 1-27, and a C-terminal fragment (ProGRP-Cfrag) which is a digest of ProGRP having an amino acid sequence from the 31st amino acid and the rest, and having no gastrin secretion promoting activity.
Holst et al. (Non-Patent Document 3) reported that in a radioimmunoassay (RIA) method using an anti-serum directed against a peptide having an amino acid sequence consisting of amino acids 42-53 of the amino acid sequence of ProGRP (hereinafter, referred to as ProGRP (42-53)), the level of ProGRP or ProGRP-Cfrag in plasma of the patients with small-cell lung carcinoma was high. However, in this method, precipitation and extraction processes were needed, and the sensitivity was insufficient. Furthermore, it is conceived that when immunization is carried out with such a short chain peptide comprising 11 amino acid residues, an antibody recognizing the conformational epitope of ProGRP is not induced.
Miyake et al. noted that ProGRP is more stable in the blood than GRP, and that an amino acid sequence consisting of amino acids 31-98, which is a common portion in the three species of ProGRP, does not show homology with the amino acid sequences of other proteins, and established a highly sensitive RIA method which does not need any precipitation and extraction processes, using an anti-serum of high titer obtained by using a recombinant peptide comprising the same amino acid sequence (hereinafter, referred to as ProGRP (31-98)) as an antigen (Non-Patent Document 1). In this method, it was shown that ProGRP served as an excellent tumor marker, in the same manner as GRP does.
However, although this method is advantageous in not needing extraction processes, measurement requires 4 days, and the sensitivity is only 10 pM (77.3 pg of antigen/mL), which is insufficient. Therefore, it is impossible to measure the ProGRP level in the serum of a normal person, and this method has not yet been developed to be clinically applied.
Furthermore, since the RIA methods of Holst et al. and Miyake et al. as described above are inhibition methods, measurement would be possible if only a portion of a fragment of ProGRP has antigenicity. But, the sensitivity is lower than that of sandwich methods, and clinical application of any ProGRP measuring method requiring high sensitivity is difficult. Thus, in order to clinically perform detection of ProGRP, it is essential to increase the sensitivity, and particularly, an antibody which can be used in sandwich methods is needed.
Yamaguchi, Aoyagi et al. developed, for the purpose of clinically applying ProGRP as a tumor marker for small-cell lung carcinoma, a convenient and highly sensitive reagent for measuring ProGRP using a sandwich method, which reagent is based on the principles of enzyme-linked immunosorbent assay (ELISA) (Patent Document 1). This assay gives results in about 2 hours, and shows high sensitivity (2 pg/mL). Thus, the assay is at present widely used in clinical applications, and it is obvious that this assay shows higher sensitivity and specificity compared to the assay using NSE with respect to small-cell lung carcinoma.
It was also found that by using this assay, the serum ProGRP values increased in neuroendocrine tumors (thyroid medullary carcinoma, etc.), and cancers exhibiting characteristics of neuroendocrine tumor (esophageal small cell carcinoma, pancreatic small cell carcinoma, prostate small cell carcinoma, etc.) as well as in small-cell lung carcinoma. Thus, it is believed that the ProGRP assay can be applied to early discovery or to the monitoring of treatment of patients with these tumors thereof.
However, although the stability of ProGRP in blood is higher than that of GRP, more fluctuation in the measured values is observed as compared to other common tumor markers. For this reason, there is a restriction in the method of using ProGRP as the object of detection, that the test sample for measurement must be frozen immediately after blood collection and stored until the time of measurement (Non-Patent Document 4).
[Patent Document 1] Japanese Patent No. 3210994
[Patent Document 2] Japanese Patent Application Laid-open No. 6-98794
[Non-Patent Document 1] Cancer Research, Vol. 54, pp. 2136-2140 (1994)
[Non-Patent Document 2] Spindel et al., Mol. Endocrinol., Vol. 1, pp. 224-232 (1987)
[Non-Patent Document 3] Holst, J. Clin. Oncol., Vol. 7, pp. 1831-1838 (1989)
[Non-Patent Document 4] Rinsho Kensa, Vol. 39, pp. 981-986 (1995)